The breast and ovarian cancer tumor suppressor protein, BRCA1, and its obligate protein partner, BARD1 are critical to proper functioning of fundamental cellular processes that support genomic stability. To date, a single biochemical activity has been identified for BRCA1/BARD1, namely, they function together as a ubiquitin E3 ligase. Although it is believed that the ability of BRCA1/BARD1 to modify specific cellular proteins with ubiquitin is fundamental to its role as a tumor suppressor, the details and ramifications of this relationship remain to be elucidated. During the past grant period, we discovered that BRCA1/BARD1 can function with ten human ubiquitin-conjugating enzymes (E2s) and that the ultimate product generated by BRCA1/BARD1 on substrates depends on the E2 present. We also identified several new substrates, including the estrogen receptor. In the next grant period we will expand our focus to include the essential subunit, BARD1, more explicitly and will participate in a collaborative effort to develop new molecular insights and tools for another RING E3 ligase that is critical in cancers, Mdm2/MdmX. The overall goals for the next grant period are 1) elucidate structural and functional determinants of mono-ubiquitin transfer and poly-ubiquitin chain formation by BRCA1/BARD1 and its interacting E2s, 2) investigate the molecular and structural determinants of BRCA1/BARD1-substrate interactions, 3) characterize BARD1 and its interactions, and 4) identify the human E2s that interact with Mdm2/MdmX, the ligase responsible for p53 regulation in vivo. A broad experimental approach will be undertaken, including biochemical, structural, molecular biological, and cellular techniques. Results from these studies will provide new insights into BRCA1/BARD1 function and will contribute to the general understanding of protein ubiquitination.